49 research outputs found
In vivo methylation of mtDNA reveals the dynamics of proteināmtDNA interactions
To characterize the organization of mtDNAāprotein complexes (known as nucleoids) in vivo, we have probed the mtDNA surface exposure using site-specific DNA methyltransferases targeted to the mitochondria. We have observed that DNA methyltransferases have different accessibility to different sites on the mtDNA based on the levels of protein occupancy. We focused our studies on selected regions of mtDNA that are believed to be major regulatory regions involved in transcription and replication. The transcription termination region (TERM) within the tRNALeu(UUR) gene was consistently and strongly protected from methylation, suggesting frequent and high affinity binding of mitochondrial transcription termination factor 1 (mTERF1) to the site. Protection from methylation was also observed in other regions of the mtDNA, including the light and heavy strand promoters (LSP, HSP) and the origin of replication of the light strand (OL). Manipulations aiming at increasing or decreasing the levels of the mitochondrial transcription factor A (TFAM) led to decreased in vivo methylation, whereas manipulations that stimulated mtDNA replication led to increased methylation. We also analyzed the effect of ATAD3 and oxidative stress in mtDNA exposure. Our data provide a map of human mtDNA accessibility and demonstrate that nucleoids are dynamically associated with proteins
Solutions of ionic liquids with diverse aliphatic and aromatic solutes ā Phase behavior and potentials for applications:A review article
This article principally reviews our research related to liquidāliquid and solidāliquid phase behavior of imidazolium- and phosphonium-based ionic liquids, mainly having bistriflamide ([NTf2]ā) or triflate ([OTf]ā) anions, with several aliphatic and aromatic solutes (target molecules). The latter include: (i) diols and triols: 1,2-propanediol, 1,3-propanediol and glycerol; (ii) polymer poly(ethylene glycol) (PEG): average molecular mass 200, 400 and 2050 ā PEG200 (liquid), PEG400 (liquid) and PEG2050 (solid), respectively; (iii) polar aromatic compounds: nicotine, aniline, phenolic acids (vanillic, ferulic and caffeic acid,), thymol and caffeine and (iv) non-polar aromatic compounds (benzene, toluene, p-xylene). In these studies, the effects of the cation and anion, cation alkyl chain and PEG chain lengths on the observed phase behaviors were scrutinized. Thus, one of the major observations is that the anion ā bistriflamide/triflate ā selection usually had strong, sometimes really remarkable effects on the solvent abilities of the studied ionic liquids. Namely, in the case of the hydrogen-bonding solutes, the ionic liquids with the triflate anion generally exhibited substantially higher solubility than those having the bistriflamide anion. Nevertheless, with the aromatic compounds the situation was the opposite ā in most of the cases it was the bistriflamide anion that favoured solubility. Moreover, our other studies confirmed the ability of PEG to dissolve both polar and non-polar aromatic compounds. Therefore, two general possibilities of application of alternative, environmentally acceptable, solvents of tuneable solvent properties appeared. One is to use homogeneous mixtures of two ionic liquids having [NTf2]ā and [OTf]ā anions as mixed solvents. The other, however, envisages the application of homogeneous and heterogeneous (PEG + ionic liquid) solutions as tuneable solvents for aromatic solutes. Such mixed solvents have potential applications in separation of the aforesaid target molecules from their aqueous solutions or in extraction from original matrices. From the fundamental point of view the phase equilibrium studies reviewed herein and the diversity of the pure compounds ā ionic liquids and target molecules ā represent a good base for the discussion of interactions between the molecules that exist in the studied solutions
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Probing Mitochondrial DNA Structure with Mitochondria-Targeted DNA Methyltransferases
The mitochondria contain their own genome, which is organized in a dynamic high-order nucleoid structure consisting of several copies of mitochondrial DNA (mtDNA) molecules associated with proteins. The mitochondrial nucleoids are the units of mtDNA inheritance, and are the sites of mtDNA transcription, replication and maintenance. Therefore, the integrity of mitochondrial nucleoids is a key determinant of mitochondrial metabolism and the bioenergetic state of the cell. Deciphering the interaction of mtDNA with proteins in nucleoprotein complexes is fundamental to understand the mechanisms of mtDNA segregation leading to mitochondrial dysfunction and to develop therapies to treat diseases associated with mtDNA mutations. The work presented in this dissertation provides essential insights into the dynamics of mtDNA interaction with nucleoid proteins. In order to unveil the organization of the mitochondrial genome, we have mapped major regulatory regions of the mtDNA in vivo using mitochondrial-targeted DNA methyltransferases. In chapter 2, we have demonstrated that DNA methyltranferases are powerful tools in probing mtDNA-protein interactions in living cells. The DNA methyltransferases\u27 accessibility to their cognate sites in the mtDNA is negatively correlated with the frequency and binding strength that protein factors occupy a specific site. Our results show that the transcription termination region (TERM) within the tRNALeu(UUR) gene is consistently and strongly protected from methylation, suggesting frequent and high affinity binding of mTERF1 (mitochondrial transcription termination factor 1). DNA methyltransferases have also been shown to be effective in detecting changes in mitochondrial nucleoid architecture due to nucleoid remodeling. We were able to determine changes in the packaging state of mitochondrial nucleoids by monitoring changes in mtDNA accessibility. The impact of altered levels of major nucleoid proteins was assessed by monitoring changes in mtDNA methylation pattern. We observed a more condensed nucleoid state causing a decrease in mtDNA methylation when the levels of the mitochondrial transcription factor A (TFAM) were altered. Changes in mtDNA methylation pattern were also evident when cells were treated with ethidium bromide (EtBr) and hydrogen peroxide. The mtDNA nucleoids adopted a less compact state during rapid mtDNA replication after EtBr treatment. In contrast, we observed a more compact mtDNA, less accessible to DNA methyltransferase after hydrogen peroxide treatment. Our results indicate that mitochondrial nucleoids are not static, but are constantly been modulated in response to factors that affect the nucleoid environment. In chapter 3, we identified the in vivo DNA binding sites of major transcription regulatory proteins, TFAM and mTERF3 using a targeted gene methylation (TAGM) strategy. In this approach, the mtDNA binding protein is fused to a DNA methyltransferase as an attempt to selectively methylate the sites adjacent to the protein target DNA region. Knowledge on how proteins interact with the mtDNA in high-order structures, which function as a mitochondrial genetic unit, will help elucidate the segregation and accumulation of mutated mtDNA in diseased tissues
The role of PGC-1 coactivators in aging skeletal muscle and heart
Aging is the progressive decline in cellular, tissue, and organ function. This complex process often manifests as loss of muscular strength, cardiovascular function, and cognitive ability. Mitochondrial dysfunction and decreased mitochondrial biogenesis are believed to participate in metabolic abnormalities and loss of organ function, which will eventually contribute to aging and decreased lifespan. In this review, we discuss what is currently known about mitochondrial dysfunction in the aging skeletal muscle and heart. We focused our discussion on the role of PGC-1 coactivators in the regulation of mitochondrial biogenesis and function and possible therapeutic benefits of increased mitochondrial biogenesis in compensating for mitochondrial dysfunction and circumventing aging and aging-related diseases
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The human motor neuron axonal transcriptome is enriched for transcripts related to mitochondrial function and microtubule-based axonal transport
Local axonal translation of specific mRNA species plays an important role in axon maintenance, plasticity during development and recovery from injury. Recently, disrupted axonal mRNA transport and translation have been linked to neurodegenerative disorders. To identify mRNA species that are actively transported to axons and play an important role in axonal physiology, we mapped the axonal transcriptome of human induced pluripotent stem cell (iPSC)-derived motor neurons using permeable inserts to obtain large amounts of enriched axonal material for RNA isolation and sequencing. Motor neurons from healthy subjects were used to determine differences in gene expression profiles between neuronal somatodendritic and axonal compartments. Our results demonstrate that several transcripts were enriched in either the axon or neuronal bodies. Gene ontology analysis demonstrated enrichment in the axonal compartment for transcripts associated with mitochondrial electron transport, microtubule-based axonal transport and ER-associated protein catabolism. These results suggest that local translation of mRNAs is required to meet the high-energy demand of axons and to support microtubule-based axonal transport. Interestingly, several transcripts related to human genetic disorders associated with axonal degeneration (inherited axonopathies) were identified among the mRNA species enriched in motor axons.
ā¢We mapped the axonal transcriptome of human spinal motor neurons.ā¢Transcripts related to mitochondrial and microtubule-based transport were enriched.ā¢These findings are consistent with results of studies in other mammalian species.ā¢Several disease-associated genes were identified among the enriched transcripts.ā¢These results underscore specific translational requirements of long motor axons
Characterization of the mitofusin 2 R94W mutation in a knockāin mouse model
CharcotāMarieāTooth disease (CMT) comprises a group of heterogeneous peripheral axonopathies affecting 1 in 2,500 individuals. As mutations in several genes cause axonal degeneration in CMT type 2, mutations in mitofusin 2 (MFN2) account for approximately 90% of the most severe cases, making it the most common cause of inherited peripheral axonal degeneration. MFN2 is an integral mitochondrial outer membrane protein that plays a major role in mitochondrial fusion and motility; yet the mechanism by which dominant mutations in this protein lead to neurodegeneration is still not fully understood. Furthermore, future preāclinical drug trials will be in need of validated rodent models. We have generated a Mfn2 knockāin mouse model expressing Mfn2R94W, which was originally identified in CMT patients. We have performed behavioral, morphological, and biochemical studies to investigate the consequences of this mutation. Homozygous inheritance leads to premature death at P1, as well as mitochondrial dysfunction, including increased mitochondrial fragmentation in mouse embryonic fibroblasts and decreased ATP levels in newborn brains. Mfn2R94W heterozygous mice show histopathology and ageādependent openāfield test abnormalities, which support a mild peripheral neuropathy. Although behavior does not mimic the severity of the human disease phenotype, this mouse can provide useful tissues for studying molecular pathways associated with MFN2 point mutations
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Expanding PRDX3 disease: broad range of onset age and infratentorial MRI signal changes
POLG mutations presenting as Charcot-Marie-Tooth disease
We report on two patients, with different POLG mutations, in whom axonal neuropathy dominated the clinical picture. One patient presented with late onset sensory axonal neuropathy caused by a homozygous c.2243G>C (p.Trp748Ser) mutation that resulted from uniparental disomy of the long arm of chromosome 15. The other patient had a complex phenotype that included early onset axonal Charcot-Marie-Tooth disease (CMT) caused by compound heterozygous c.926G>A (p.Arg309His) and c.2209G>C (p.Gly737Arg) mutations